PROJECT SUMMARY/ABSTRACT Malignant melanoma incidence is rapidly increasing, requiring new detection and therapeutic approaches. Despite the development of targeted therapies, mortality rates of advanced melanoma remain stubbornly high. If detected in its earliest stages, however, melanoma can often be cured. Activating V600E mutations in the BRAF proto-oncogene drive approximately 50% of all cutaneous melanomas. Yet, when a melanocyte (the melanoma cell of origin) acquires a BRAFV600E mutation, the cell does not immediately transition to malignancy. Instead, it undergoes rapid proliferation followed by growth arrest resulting in a stable pigmented skin macule known as a benign nevus or mole. Few moles ever progress to melanoma suggesting that nevus cells have a robust intrinsic defense against hyperproliferation. The mechanisms underlying BRAFV600E nevus formation, however, remain elusive. To address this critical gap in our understanding of intrinsic barriers to melanoma progression, I propose to investigate factors controlling BRAFV600E-driven growth arrest. Addressing this unmet need will deepen our understanding of the events initiating melanoma while improving our ability to detect nevi at risk for progression to melanoma. DNA sequencing of human nevi revealed that additional genetic changes do not distinguish proliferating BRAFV600E melanocytes from the growth-arrested melanocytes of the nevus, suggesting that epigenetic elements may restrain hyperproliferation. Preliminary data from our lab shows that microRNAs miR-211-5p and miR-328-3p are upregulated in growth arrested BRAFV600E nevi compared to both normal melanocytes and to melanomas arising from adjacent benign nevi. Expression of each microRNA in human melanocytes leads to a growth-arrest phenotype suggesting miRs 211-5p and 328-3p play a functional role in establishing the growth- arrested state of nevi. Moreover, both microRNAs regulate the mRNA transcripts of YWHAZ, a gene whose upregulation in correlated with cancer progression and whose downregulation leads triggers cell cycle arrest. Accordingly, we will investigate the roles of miR-211-5p and miR-328-3p in BRAFV600E-driven nevus formation. Using CRISPR-mediated genome engineering, synthetic microRNAs, RNA sequencing, and human cells derived directly from patient nevi, we will specifically determine whether miRs 211-5p and 328-3p deplete YWHAZ mRNA in BRAFV600E melanocytes leading to growth arrest and serving as a barrier to melanoma. Successful completion of this proposal will yield new insight into the cell-intrinsic mechanisms that enable melanocytes to resist BRAFV600E-driven malignant transformation, and further probe how nevi form. Moreover, this proposal will further our understanding of the key genetic and epigenetic events initiating melanoma while improving our ability to detect the disease at its earliest time points.